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Background: Extracellular vesicles (EVs) are essential for cell-to-cell communication because they transport functionally active molecules, including proteins, RNA, and lipids, from secretory cells to nearby or distant target cells. Seminal plasma contains a large number of EVs (sEVs) that are phenotypically heterogeneous. The aim of the present study was to identify the RNA species contained in two subsets of porcine sEVs of different sizes, namely small sEVs (S-sEVs) and large sEVs (L-sEVs). The two subsets of sEVs were isolated from 54 seminal plasma samples by a method combining serial centrifugations, size exclusion chromatography, and ultrafiltration. The sEVs were characterized using an orthogonal approach. Analysis of RNA content and quantification were performed using RNA-seq analysis. Results: The two subsets of sEVs had different size distributions (P < 0.001). They also showed differences in concentration, morphology, and specific protein markers (P < 0.05). A total of 735 RNAs were identified and quantified, which included: (1) mRNAs, rRNAs, snoRNAs, snRNAs, tRNAs, other ncRNAs (termed as "all RNAs"), (2) miRNAs and (3) piRNAs. The distribution pattern of these RNA classes differed between S-sEVs and L-sEVs (P < 0.05). More than half of "all RNAs", miRNAs and piRNAs were found to be differentially abundant between S- and L-sEVs (FDR < 0.1%). Among the differentially abundant RNAs, "all RNAs" were more abundant in L- than in S-sEVs, whereas the most of the miRNAs were more abundant in S- than in L-sEVs. Differentially abundant piRNAs were equally distributed between S- and L-sEVs. Some of the all RNAs and miRNAs found to be differentially abundant between S- and L-sEVs were associated with sperm quality and functionality and male fertility success. Conclusions: Small and large sEVs isolated from porcine seminal plasma show quantitative differences in RNA content. These differences would suggest that each sEV subtype exerts different functional activities in the targeted cells, namely spermatozoa and functional cells of the female reproductive tract. [ABSTRACT FROM AUTHOR]

Background: To address knowledge gaps, this study aimed to investigate the involvement of inflammasomes in the etiology of azoospermia. This study focused on the gene expression of key inflammasome components, including NLR family pyrin domain containing 3 (NLRP-3), CASPASE-1, Interleukin-1β (IL-1β), Interleukin-18 (IL-18), NLR family CARD domain-containing protein 4/ice protease-activating factor (NLRC-4/IPAF) , and Absent in melanoma 2 (AIM-2). Methods: We analyzed gene expression in blood and testicular tissue from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). Additionally, we compared IL-1β and IL-18 expression levels in seminal plasma samples using the Enzyme-Linked Immunosorbent Assay (ELISA) method. For comparison, blood samples from normospermic (NS) individuals were also genetically evaluated. Results: Our results indicated significantly higher gene expression of inflammasome components in NOA patients than those in OA patients either in blood or in testicular tissue. Both azoospermic groups exhibited higher mRNA levels of inflammasome genes comparing with those from blood samples of NS men. Seminal plasma samples showed significantly increased levels of IL-1β and IL-18 in NOA patients compared to men with OA. The ROC curve analysis indicated strong and significant predictive power of IL-18, AIM-2 and NLRC-4/IPAF gene expression profiles between NOA vs. NS patients and NOA vs. OA. Conclusions: Our findings highlight the role of hidden chronic inflammation in azoospermia, particularly within the NOA group. This study provides a foundation for further detailed research, which could aid in the development of diagnostic panels to differentiate between various azoospermic groups. [ABSTRACT FROM AUTHOR]

Sexual dimorphism involves distinct anatomical, physiological, behavioral, and developmental differences between males and females of the same species, influenced by factors prior to conception and during early development. These sex-specific traits contribute to varied phenotypes and individual disease risks within and across generations and understanding them is essential in mammalian studies. Hormones, sex chromosomes, and imprinted genes drive this dimorphism, with over half of quantitative traits in wildtype mice showing sex-based variation. This review focuses on the impact of paternal non-genetic factors on sexual dimorphism. We synthesize current research on how paternal health before conception affects offspring phenotypes in a sex-specific manner, examining mechanisms such as DNA methylation, paternally imprinted genes, sperm RNA, and seminal plasma. Additionally, we explore how paternal influences indirectly shape offspring through maternal behavior, uterine environment, and placental changes, affecting males and females differently. We propose mechanisms modulating sexual dimorphism during development, underscoring the need for sex-specific documentation in animal studies. [ABSTRACT FROM AUTHOR]

Prolactin induced-protein (PIP) has been found to be rich in immunomodulatory epitopes, including N-acetyllactosamine (LacNAc) and N,N-diacetyllactosamine (LacdiNAc) residues, which may constitute ligands for galecin-3 (Gal-3). In the current study, we aimed to investigate the reactivity of galactose- and N-acetylgalactosamine-specific lectins with human seminal plasma PIP. Subsequently, we examined the direct interaction between seminal plasma PIP and galectin-3, and next analyzed whether there are any differences in the interaction associated with impaired semen parameters. The reactivity of terminal galactose-presenting glycans in seminal plasma PIP with Ricinus communis agglutinin I in the asthenozoospermic group was significantly higher compared to the normozoospermic fertile subjects. Investigating the reactivity of Wisteria floribunda lectin with PIP glycans, we found likewise significantly higher relative reactivity in the normozoospermic infertile as well as the oligoasthenozoopermic group compared to the control group. These results are related to the expression of LacdiNAc epitopes in the oligosaccharide chain of PIP. Finally, we observed that PIP reactivity with Wisteria floribunda lectin correlates positively with the interaction between galectin-3 and PIP in the seminal plasma. This can suggest that LacdiNAc residues are engaged in the interaction between PIP and galectin-3. [ABSTRACT FROM AUTHOR]

Simple Summary: Seminal plasma (SP) is a crucial component of semen that significantly influences sperm function. However, the relationship between SP and the freezability of boar sperm remains underexplored compared to the functional diversity of SP. Utilizing metabolomics and proteomics approaches, we identified 13 differentially expressed metabolites, predominantly lipids such as phosphoethanolamine, and 38 proteins including CRYAA, CUTC, SHANK1, PFN1, NEU1, SAA3, TACSTD2, APOA2, and CCN6. These metabolites and proteins were enriched in processes and functions related to cytoskeleton dynamics and cell adhesion. Notably, 33 metabolites showed significant correlations with the average progressive motility (PM) at 10 min and 2 h post-thawing. Among these, seven negatively correlated metabolites, including myrisamine oxide and minoxidil, were identified as drugs or environmental pollutants, while positively correlated metabolites included glycerol phosphocholine, creatine, and carnitine. Furthermore, we identified 70 related proteins enriched in gene ontology (GO) terms associated with cell division and cycle regulation, as well as KEGG pathways involving thermogenesis and pyruvate metabolism. The metabolites and proteins linked to average PM at 10 min and 2 h post-thawing were jointly enriched in pathways related to thermogenesis, arginine and proline metabolism, and ether lipid metabolism. Additionally, six reproductive hormones, including testosterone and estradiol, were detected in SP using ELISA; however, none showed significant correlations with semen quality before or after freezing. When using highly and lowly freezable SP as base freezing extenders, the protective effect of highly freezable SP was not significantly superior to that of lowly freezable SP, and it did not outperform the control group using a commercial freezing extender. Background: Seminal plasma is an important component of semen and has a significant effect on sperm function. However, the relationship between seminal plasma and sperm freezing capacity has not been fully studied. Purpose: Exploring metabolites and proteins related to the boar sperm freezing capacity in seminal plasma, by metabolomic and proteomic approaches, and directly verifying the protective effect of seminal plasma on the cryopreservation of boar sperm using high and low freezability seminal plasma as base freezing extender. Methods: Semen samples were collected from 30 different boars, 11 high and 11 low freezing-resistant boars were selected after freezing 2~4 times, and seminal plasma was selected at the same time. Sperm motility and movement parameters were analyzed using a CASA system. Reproductive hormones (Testosterone, progesterone, estradiol, prolactin, prostaglandin F2α, luteinoid hormone) in seminal plasma were detected by ELISA. Analysis of proteins and metabolites in high and low freezing-resistant seminal plasma by proteomics and metabolomics techniques. Results: The six reproductive hormones tested were not significantly associated with sperm freezing resistance. A total of 13 differentially expressed metabolites (DEMs) and 38 differentially expressed proteins (DEPs) were identified, while a total of 348 metabolites and 1000 proteins were identified. These DEMs were related to energy metabolism, drugs, or environmental pollutants, while the DEPs were mainly involved in the cytoskeletal dynamics and cell adhesion processes. There were 33 metabolites and 70 proteins significantly associated with mean progress motility (PM) at 10 min and 2 h after thawing. The 70 related proteins were associated with cell division and cycle regulation in gene ontology (GO) terms, as well as KEGG pathways, thermogeneration, and pyruvate metabolism. Using highly freezable boar SP as a base freezing extender made no difference from using lowly freezable boar SP, and both were not as good as the commercial control. Conclusion: There were significant differences in seminal plasma with different freezability, but the similarity was much greater than the difference. The protection effect of seminal plasma is not remarkable, and it does not exhibit superior cryoprotective properties compared to commercial semen cryoelongators. Significance: This study provides a deeper understanding of how seminal plasma composition affects sperm freezabilty. It provides potential biomarkers and targets for improving sperm cryopreservation techniques. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, 25 rams were randomly divided into five groups to investigate the effects of different photoperiods on the ram sperm. The control group was exposed to 12 h of light, while the experimental groups were exposed to 14, 16, 18, and 20 h of light. The results showed that compared to the control group, the group exposed to 16 h of light had significantly improved sperm motilities, sperm concentrations, ejaculate volumes, total sperm counts, sperm abnormalities, acrosome integrities, and membrane integrities (p < 0.05). Additionally, we identified 345 different metabolites between the control and 16 h light group, with 273 upregulated and 72 downregulated. The amino acid content of the seminal plasma was significantly higher in the 16 h light group compared to the control (p < 0.05). Furthermore, compared to the control group, the 16 h light group exhibited significantly higher levels of seminal plasma testosterone, serum follicle-stimulating hormone (FSH), and luteinizing hormone (LH) (p < 0.05). In terms of the sperm antioxidant capacity, the catalase (CAT) activity was the highest in the 16 h light group. Additionally, prolonged light exposure between 14 and 18 h increased the glutathione (GSH) levels (p < 0.05), and the malondialdehyde (MDA) levels reached their lowest point at 16 h of light exposure (p < 0.05) but increased again with 20 h of light. Overall, the artificial extension of the photoperiod to 16 h had a positive effect on the ram sperm quality. This investigation aimed to study the effects of varying light exposure durations on ram sperm. A total of 25 rams were randomly divided into five groups. The control group was exposed to light durations of 12 h, while the experimental groups were exposed to light durations of 14, 16, 18, and 20 h. After three months of rearing, semen was collected from each ram four times using the artificial vagina method. The sperm motility parameters, sperm abnormality, sperm concentration, acrosome integrity, membrane integrity, semen volume, and total sperm number were measured. Thereafter, the metabolome, amino acid level, testosterone content, plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, and sperm antioxidant capacity were measured. The results showed that the sperm motility, sperm concentration, ejaculation volume, total sperm number, acrosome integrity, and membrane integrity in the 16 h light group were significantly improved compared to the control (p < 0.05), meanwhile the sperm abnormality was decreased. Moreover, we found 345 different metabolites between the control and 16 h light group. Among these, 273 were upregulated and 72 were downregulated. Furthermore, the amino acid content of the seminal plasma in the 16 h light group was significantly increased (p < 0.05) compared to the control. Interestingly, the seminal plasma testosterone content and the levels of FSH and LH in the serum in the 16 h light group were significantly increased (p < 0.05) compared to the control. In terms of the sperm antioxidant capacity, it was observed that the CAT activity was the highest in the group exposed to 16 h of light and decreased at 18 h of light exposure when compared to the control group; however, the CAT activity at 20 h was not different from the control. Additionally, within the 14 to 18 h light exposure range, prolonged light exposure increased the GSH content (p < 0.05), whereas 20 h of light exposure reduced the GSH content. The MDA levels decreased with prolonged light exposure, reaching the lowest point at 16 h (p < 0.05), but increased again at 20 h of light exposure. KEGG analysis indicated that the differential metabolites were mainly involved in metabolic and synthetic activities. Based on the results of this study, we can conclude that the artificial extension of the light duration for 16 h has a positive effect on ram sperm quality. [ABSTRACT FROM AUTHOR]

Background/Objectives: Varicoceles are a common contributor to male infertility, significantly impacting male-factor infertility cases. Traditional diagnostic methods often lack the sensitivity to detect the molecular and cellular disruptions caused by varicoceles, limiting the development of effective, personalized treatments. This narrative review aims to explore the advancements in proteomics and metabolomics as innovative, non-invasive diagnostic tools for varicocele-associated male infertility and their potential in guiding personalized therapeutic strategies. Methods: A comprehensive literature search was conducted using databases such as PubMed, Scopus, and Web of Science up to October 2024. Studies focusing on the application of proteomic and metabolomic analyses in varicocele-associated male infertility were selected. The findings were critically analyzed to synthesize current knowledge and identify future research directions. Results: Proteomic analyses revealed differentially expressed proteins in the sperm and seminal plasma of varicocele patients, revealing disruptions in pathways related to oxidative stress, mitochondrial dysfunction, apoptosis, and energy metabolism. Key proteins such as heat shock proteins, mitochondrial enzymes, and apoptotic regulators were notably altered. Metabolomic profiling uncovered specific metabolites in seminal plasma—such as decreased levels of lysine, valine, and fructose—that correlate with impaired sperm function and fertility potential. The integration of proteomic and metabolomic data provides a comprehensive molecular fingerprint of varicocele-induced infertility, facilitating the identification of novel biomarkers for early diagnosis and the development of personalized therapeutic interventions. Conclusions: Advances in proteomics and metabolomics have significantly enhanced our understanding of the molecular mechanisms underlying varicocele-associated male infertility. These "omics" technologies hold great promise for improving diagnostic accuracy and personalizing treatment, ultimately leading to better outcomes for affected men. Future large-scale clinical trials and validations are essential to confirm these biomarkers and facilitate their integration into routine clinical practice. [ABSTRACT FROM AUTHOR]

Background/Objectives: Non-obstructive azoospermia (NOA) is a severe form of male infertility characterized by the absence of sperm in the ejaculate due to impaired spermatogenesis. Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection is the primary treatment, but success rates are unpredictable, causing significant emotional and financial burdens. Traditional clinical and hormonal predictors have shown inconsistent reliability. This review aims to evaluate current and emerging non-invasive preoperative predictors of successful sperm retrieval in men with NOA, highlighting promising biomarkers and their potential clinical applications. Methods: A comprehensive literature review was conducted, examining studies on clinical and hormonal factors, imaging techniques, molecular biology biomarkers, and genetic testing related to TESE outcomes in NOA patients. The potential role of artificial intelligence and machine learning in enhancing predictive models was also explored. Results: Traditional predictors such as patient age, body mass index, infertility duration, testicular volume, and serum hormone levels (follicle-stimulating hormone, luteinizing hormone, inhibin B) have limited predictive value for TESE success. Emerging non-invasive biomarkers—including anti-Müllerian hormone levels, inhibin B to anti-Müllerian hormone ratio, specific microRNAs, long non-coding RNAs, circular RNAs, and germ-cell-specific proteins like TEX101—show promise in predicting successful sperm retrieval. Advanced imaging techniques like high-frequency ultrasound and functional magnetic resonance imaging offer potential but require further validation. Integrating molecular biomarkers with artificial intelligence and machine learning algorithms may enhance predictive accuracy. Conclusions: Predicting TESE outcomes in men with NOA remains challenging using conventional clinical and hormonal parameters. Emerging non-invasive biomarkers offer significant potential to improve predictive models but require validation through large-scale studies. Incorporating artificial intelligence and machine learning could further refine predictive accuracy, aiding clinical decision-making and improving patient counseling and treatment strategies in NOA. [ABSTRACT FROM AUTHOR]

Three different centrifugation frequencies viz., zero time (ZTC), one time (ITW), two times (2TC) and three times (3TC) supplemented with different levels of vitamin E were studied under in vitro cryosurvival of West African Dwarf (WAD) goat spermatozoa. Semen samples were washed ITW, 2TC and 3TC and supplemented each with 2, 4, 6 and 8 mM of vitamin E in a 3x4 factorial arrangements and cryopreserved for 30 days in liquid nitrogen. Data were subjected to one-way ANOVA. Spermatozoa cryopreserved with Tris-based extender supplemented with 6 and 8 mM using 3TC and 8 mM with 2TC had highest (P<0.05) percentage motility, percentage acrosome integrity and percentage live spermatozoa while highest (P<0.05) percentage membrane integrity were observed at 4, 6 and 8 mM. Cryopreserved spermatozoa with 6 and 8 mM for 3TC had the lowest (P<0.05) percentage abnormality and highest (P<0.05) percentage spermatozoa that underwent acrosome reaction including 8 mM using 2TC. Also 4, 6 and 8 mM for 3TC and 6 and 8 mM using 2TC had the highest (P<0.05) percentage spermatozoa that underwent capacitation. The results showed lowest (P < 0.05) concentrations of Malondialdehyde MDA at 2, 4, 6 and 8 mM for 1TC, 6 and 8 mM for 2TC and ZTC; and 8 mM vitamin E for 3TC. However, the Tris extenders supplemented with 2 and 4 mM for ZTC and 4 mM for 1TC had highest (P<0.05) values for arginase activity. Cryopreservation of WAD goat buck sperm with 2TC and 3TC improved viability and fertilizing ability. [ABSTRACT FROM AUTHOR]

Three different centrifugation frequencies viz., zero time (ZTC), one time (ITW), two times (2TC) and three times (3TC) supplemented with different levels of vitamin E were studied under in vitro cryosurvival of West African Dwarf (WAD) goat spermatozoa. Semen samples were washed ITW, 2TC and 3TC and supplemented each with 2, 4, 6 and 8 mM of vitamin E in a 3x4 factorial arrangements and cryopreserved for 30 days in liquid nitrogen. Data were subjected to one-way ANOVA. Spermatozoa cryopreserved with Tris-based extender supplemented with 6 and 8 mM using 3TC and 8 mM with 2TC had highest (P

Abstract Background Male infertility is a major problem for many couples in the world. Many factors could cause male infertility such as environmental and genetic factors, life style, aging, inflammation, endocrinological etiologies, and antisperm antibodies. Main body Circulating cell-free nucleic acids (cfNAs) may play a key role in male infertility. cfNAs are obtained from different body fluids such as blood plasma, cerebrospinal fluid, amniotic fluid, urine, bronchoalveolar lavage fluid, and seminal plasma. The different types of cfNAs present in human semen include cell-free DNAs, cell free RNAs and cell-free mitochondrial DNAs and they are differentially higher than those in other body fluids. Few evidence have been done regarding the direct relationship between cfNAs and male infertility in serum and seminal plasma of infertile men compared to the fertile men. Conclusions This document aimed to compile data about the main causes influencing male infertility focusing on seminal cfNA/cfDNA and its possible role as differential biomarker to diagnosis the main source of spermatogenesis abnormalities and male infertility.

Abstract Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

Despite the significant progress in sperm cryopreservation methods in recent years, research has proven that long-term storage of sperm in freezing and cold storage causes serious damage to sperm. The occurrence of fat peroxidation and the metabolism of spermatozoids results in destruction of spermatozoa during freezing and storage. Temperature shock, the formation of intracellular ice crystals, cellular dehydration, increased salt concentration and osmotic shock may occur during the freezing and thawing process. In addition, cryopreservation causes harmful structural changes in the plasma membrane. Changes in membrane permeability to some ions such as calcium during the freezing and thawing process have been reported. Also, during cryopreservation, the formation of intracellular and extracellular ice causes cell destruction and death. The sperm plasma membrane is the main site of damage during the freezing and thawing process and is very sensitive to lipid peroxidation due to the presence of many unsaturated fatty acids. These changes may have a role in the accumulation of toxic products of metabolism, mainly reactive oxygen species (ROS) that are generated through lipid peroxidation of sperm membranes. Small amounts of ROS play an important role in sperm capacitation, increased sperm motility, acrosome reaction, and oocyte fertilization in humans, cows, horses, pigs, and rams. Therefore, to solve these problems, various antioxidants, preservatives and chelators are used in sperm extenders.

Several seminal plasma components, besides NGF, are implicated as ovulation-inducing factors in mammals. This study investigated the IL1B and its receptor IL1R1 in the testis (T), male accessory glands, prostate (P) and seminal vesicles (SV), and uterus (U) of adult rabbits using immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). We also assessed the presence of IL1B in seminal plasma through Western blotting (WB) and examined the interaction between IL1B and NGF in vitro by measuring their production with enzyme-linked immunosorbent assay (ELISA) in the presence of NGF and IL1B alone or with their respective receptor antagonists. IHC revealed IL1B system expression in all reproductive organs studied, with IL1B and IL1R1 localized to the germinative epithelium of the T and the epithelial cells of the accessory glands and U. IL1B gene transcript levels were significantly higher (p < 0.01) in the P and SV compared to the T, while IL1R1 levels were significantly higher (p < 0.001) in the P compared to the other tissues, while IL1R1 levels were three times higher (p < 0.001) in the P. WB confirmed the presence of IL1B in seminal plasma with a 30–35 kDa band. The in vitro study demonstrated that IL1B increased (p < 0.05) basal NGF production in the U, whereas NGF had no effect on IL1B production. These findings provide evidence of the expression of the IL1B/IL1R1 system in both male and female rabbit reproductive tracts and suggest that IL1B in seminal plasma may influence uterine endocrine activity. The results propose a potential role for IL1B in ovulation, in conjunction with NGF, supporting that ovulation may involve inflammatory-like processes. [ABSTRACT FROM AUTHOR]

Giant pandas are an endangered species with low reproductive rates. Cryptorchidism, which can negatively affect reproduction, is also often found in pandas. Seminal plasma plays a crucial role in sperm–environment interactions, and its properties are closely linked to conception potential in both natural and assisted reproduction. The research sought to identify seminal fluid protein content variations between normal and cryptorchid giant pandas. Methods: Using a label-free MS-based method, the semen proteomes of one panda with cryptorchidism and three normal pandas were studied, and the identified proteins were compared and functionally analyzed. Results: Mass spectrometry identified 2059 seminal plasma proteins, with 361 differentially expressed proteins (DEPs). Gene ontology (GO) analysis revealed that these DEPs are mainly involved in the phosphatecontaining compound metabolic, hydrolase activity, and kinase activity areas (p ≤ 0.05). The KEGG functional enrichment analysis revealed that the top 20 pathways were notably concentrated in the adipocyte lipolysis and insulin metabolism pathway, with a significance level of p ≤ 0.05. Further analysis through a protein–protein interaction (PPI) network identified nine key proteins that may play crucial roles, including D2GXH8 (hexokinase Fragment), D2HSQ6 (protein tyrosine phosphatase), and G1LHZ6 (Calmodulin 2). Conclusions: We suspect that the high abundance of D2HSQ6 in cryptorchid individuals is associated with metabolic pathways, especially the insulin signal pathway, as a typical proteomic feature related to its pathological features. These findings offer insight into the ex situ breeding conditions of this threatened species. [ABSTRACT FROM AUTHOR]

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. [ABSTRACT FROM AUTHOR]

We recently demonstrated the effectiveness of long-term treatment with rifaximin and the probiotic DSF (De Simone formulation) in improving urogenital and gastrointestinal symptoms in patients with both chronic inflammatory prostatitis (IIIa prostatitis) and diarrhea-predominant irritable bowel syndrome (IBS-D), relative to patients with IBS-D alone. Because the low-grade inflammation of the intestine and prostate may be one of the reasons for co-developing both IIIa prostatitis and IBS-D, we designed the present study to once again evaluate the efficacy of combined rifaximin and DSF treatment in patients affected by IIIa prostatitis plus IBS-D, but we also measured seminal plasma pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines before and after treatment. Methods: We consecutively enrolled 124 patients with IIIa prostatitis and IBS-D (diagnosed using the Rome III criteria). Patients were randomized into two groups: group A (n = 64) was treated with rifaximin (seven days per month for three months) followed by DSF, and group B (n = 60) was treated with a placebo. By the end of the intervention, 68.7% and 62.5% of patients from group A reported improved NIH-CPSI (National Institute of Health's Chronic Prostatitis Symptom Index) and IBS-SSS (Irritable Bowel Syndrome Severity Scoring System) scores, respectively, compared to only 3.3% and 5% of the placebo group. Group A patients also had significantly lower mean seminal plasma levels of IL-6 (11.3 vs. 32.4 pg/mL) and significantly higher mean levels of IL-10 (7.9 vs. 4.4 pg/mL) relative to baseline, whereas the levels of IL-6 and IL-10 did not change in the placebo group. Conclusions: The combined treatment with rifaximin and DSF appears to represent the optimal approach for addressing a syndrome such as irritable bowel syndrome (IBS-D plus), which frequently co-occurs with prostatitis (IIIa prostatitis). This approach is particularly beneficial in cases where the symptoms are not always clearly delineated, the etiology is multifactorial, and the diagnosis is multilevel. [ABSTRACT FROM AUTHOR]

Seminal plasma is rich in proteins originating from various male reproductive organs. The phosphorylation of these proteins can significantly impact sperm motility, capacitation, and acrosome reaction. Phosphoproteomics identifies, catalogues, and characterizes phosphorylated proteins. The phosphoproteomic profiling of seminal plasma offers valuable insights into the molecular mechanisms that influence semen quality and male fertility. Thus, the aim of this study was a phosphoproteomic analysis of white and yellow turkey seminal plasma. The experimental material consisted of 100 ejaculates from BIG-6 turkeys between 39 and 42 weeks of age. The collected white and yellow turkey seminal plasmas were analyzed for total protein content; the activity of selected enzymes, i.e., alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT); and the content of reduced glutathione (GSH) and malondialdehyde (MDA). Phosphoproteins were isolated from white and yellow seminal fluids, and the resulting protein fractions were separated by SDS-PAGE and Western blotting. Phosphorylated residues were immunodetected, and the isolated phosphoproteins were identified (nano LC-MS/MS). Yellow seminal plasmas were characterized by higher levels of total protein, GSH, and MDA, as well as higher levels of ALP, ACP, and GPx activity. There were no significant differences in the activity of SOD and CAT. A total of 113 phosphoproteins were identified in turkey seminal fluids. The functional analysis demonstrated that these phosphoproteins were mainly involved in oocyte fertilization, organization and metabolism of the actin cytoskeleton, amplification of the intracellular signal transduction pathway, general regulation of transport, vesicular transport, proteome composition of individual cellular compartments, and the organization and localization of selected cellular components and macromolecules. Increased phosphorylation of the fractions containing proteins encoded by SPARC, PPIB, TRFE, QSOX1, PRDX1, PRDX6, and FASN genes in white plasmas and the proteins encoded by CKB, ORM2, APOA1, SSC5D, RAP1B, CDC42, FTH, and TTH genes in yellow plasmas was observed based on differences in the optical density of selected bands. The obtained results indicate that the phosphorylation profiles of turkey seminal plasma proteins vary depending on the type of ejaculate. [ABSTRACT FROM AUTHOR]

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female. [ABSTRACT FROM AUTHOR]

Background Objectives Materials and methods Results Discussion Conclusion Within‐subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood.To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men.Semen parameters were assessed in samples collected 6–8 days apart from 164 18–19‐year old participants of the Russian Children's Study, a prospective cohort. Subsets of paired samples were used for label‐free quantitation and targeted mass‐spectrometry of proteins in seminal plasma (SP) and seminal extracellular vesicles (EVs), and for small non‐coding RNA (sncRNA) profiling in EVs and spermatozoa using RNA‐seq. The mean difference between two ejaculates, within‐subject variation, intraclass correlation, and concordance correlation were used to assess IIV for all parameters. Low variability with high reproducibility and high reliability was considered if CVw < 15% and ICC > 0.90, respectively.Analytical variability was low for all investigated parameters in technical replicates. IIV was assessed for basic semen parameters and proteins in SPs and EVs: 319 and 777 proteins, respectively, using untargeted analysis; 9 and 10 proteins using targeted quantification. We also described the IIV for sncRNA, including microRNA, piwi‐interacting RNA, tRNA, and tRNA‐derived small RNA (tsRNA) in EVs (409 sncRNA and 78 tsRNA) and in spermatozoa (265 sncRNA and 15 tsRNA). We identified 22 and 27 non‐overlapping proteins in SP and EVs, respectively, and 46 and 9 sncRNA, including 5 and 0 tsRNA in seminal EVs and spermatozoa, respectively, with low variability. The fatty acid synthase (FAS) had the lowest IIV in both media in targeted protein quantification.We identified a number of proteins and sncRNA with low variability among 111 proteins, 176 sncRNA, and 12 tsRNA which were previously suggested as biomarkers of male fertility and reproductive outcomes: lactotransferrin, cysteine‐rich secretory protein 3, alpha‐1‐antichymotrypsin, epididymal sperm‐binding protein 1, glutathione S‐transferase Mu 3, alpha‐1‐acid glycoprotein 2, serum amyloid P‐component, aminopeptidase N, neprilysin, FAS, and miR‐10b‐3p, miR‐122‐5p, miR‐205‐5p, miR‐222‐3p, miR‐34c‐5p, miR‐509‐3‐5p, miR‐888‐5p, miR‐892a, miR‐363‐3p, miR‐941, miR‐146a‐5p, miR‐744‐5p.These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health. [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effect of the winter and spring seasons on some biomarkers and semen characteristics of Holstein bulls (n = 20) during the Coronavirus pandemic period. Semen was collected for 16 weeks and evaluated weekly, along with the determination of some biomarkers. Sperm concentration, live sperm percentage, the concentrations of the most amino acids, essential, non-essential, and total amino acids, all carboxylic acids, omega 3, 6, 9, total fatty acids, and total saturated and unsaturated fatty acids, increased (P<0.05) in semen of spring compared to the winter season. Moreover, the percentage of plasma membrane integrity was higher in the winter than the spring season. The concentration of malondialdehyde in semen decreased (P<0.05) in the semen during the winter compared to the spring season. These biomarkers may have played a role in the fertility and semen quality or were affected by the season and repercussions of the Corona pandemic (feed shortage stress). In conclusion, the level of some amino and carboxylic acids can be adopted as an indicator of climate change (season) and semen quality of bulls or energy level in the diet. [ABSTRACT FROM AUTHOR]

The establishment of pregnancy involves a fine‐tuned balance between protection and tolerance within the maternal immune system, as the female needs to accept a foreign antigen (the semi‐allogenic fetus) while still being able to combat pathogens from the uterus. In the horse, the first uterine exposure to paternal antigens is during mating when sperm is introduced to the tissue and draining lymphatics of the uterus. Additionally, it has been suggested that seminal plasma and its proteins within it play an essential role in preparing the female tract for a suitable immunologic environment but this has not been confirmed in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with seminal plasma or with reduced seminal plasma. We hypothesised that reduced seminal plasma would alter the endometrial transcriptome and affect transcripts relating to immunotolerance, antigen presentation and embryo growth and development. To do so, six (n = 6) mares were inseminated in a randomised switch‐back design over the course of four oestrous cycles. Mares were rectally palpated and scanned via ultrasonography for the detection of a pre‐ovulatory follicle (>35 mm) alongside increasing uterine oedema and relaxed cervix, and then treated with one of four treatment groups including (1) 30 mL lactated Ringers solution (LRS; NegCon), (2) 500 × 106 spermatozoa in conjunction with 30 mL seminal plasma (SP+), (3) 30 mL lactated Ringers solution (LRS; wash out) and (4) 500 × 106 spermatozoa with seminal plasma reduced via gradient centrifugation and resuspended in 30 mL LRS (SP−). Human chorionic gonadotropin (hCG) was administered to standardise the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilising Trizol, and RNA‐Seq was performed by Novogene, with 97.79% total mapping and 40 million read depth. p value was set to <0.05. When comparing SP+ to SP−, 158 differentially expressed genes (DEGs) were identified. Biological processes impacted included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Gene ontology (GO) enrichment analysis using DAVID v6.8 revealed that many of these DEGs were involved in biological process such as antigen presentation (HLA‐DM beta chain, HLA‐DRB, HLA‐DQA and RASGRP1), immune cell signalling (CXCL9, CXCL1, DEFB1 and MIP‐2B), embryo growth and development (INHA, KLF2, RDH10, LAMA3 and SLC34A2) and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2 and IGFBP3). Overall, reduction of seminal plasma from the insemination dose impacted the endometrial transcriptome at the time of early embryonic exposure to the uterine environment. Further work is justified to evaluate these alterations impact on embryo maturation, placental development, pregnancy outcome and development of offspring. [ABSTRACT FROM AUTHOR]

N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N- glycopeptides in seminal plasma, we identified 92 intact N- glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility. [ABSTRACT FROM AUTHOR]

Introduction: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extendermay hindermolecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. Methods: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Results: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. Conclusion: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of somemiRNAs fromthe extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender. [ABSTRACT FROM AUTHOR]

Context: The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims: Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods: Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2 h). Key results: Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P < 0.05) and greater sperm motility index and beat cross frequency than NOSP after 2 h post-thaw incubation (P < 0.05). The addition of HSP improved sperm kinematic parameters compared to NOSP and GSP treatments (P < 0.05). Conclusions and implications: These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted. There is an urgent need to develop a means of freezing Asian elephant semen, to enable captive breeding conservation programs to maintain genetic diversity. This study revealed important information about the proteins present in the seminal plasma of these animals and how this may contribute to the successful frozen storage of their spermatozoa. These findings will contribute to our understanding of differences in sperm quality between elephants and aid in developing sperm freezing protocols that will lead to the long-awaited birth of an Asian elephant calf from frozen spermatozoa. Photograph by Cameron Negus. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

Background: Male infertility is a multifactorial condition influenced by various physiological and biochemical factors. Seminal fluid composition plays a crucial role in sperm function and fertilization potential. Chitotriosidase is a chitinase enzyme released by activated macrophages and is highly conserved and controlled. The notable chitinase in humans plays a significant role in the body's immunological response and is linked to inflammation, infection, tissue damage, and remodeling processes. On the other hand, malondialdehyde is a marker of lipid peroxidation, reflecting oxidative stress levels. Objective: This study aimed to explore the correlation between malondialdehyde levels and Chitotriosidase-1in seminal fluid in Iraqi infertile males. Methods: Ninety males aged between twenty and forty-five were included in this cross-sectional study, all diagnosed with infertility by specialists at the infertility unit of Al-Batool Teaching Hospital between February 2022 and February 2023. The participants were categorized into three groups: the Normozoospermic Group (G1), the Asthenospermia Group (G2), and the Oligozoospermic Group (G3). Seminal malondialdehyde and Chitotriosidase-1 levels were measured by competitive Enzyme-linked immunosorbent assay. Results: The study findings showed significantly higher levels of seminal fluid Chitotriosidase-1 found in the G2 group compared to the G3 and G1 groups. The seminal fluid malondialdehyde level for G1 was significantly lower than those for G2 and G3, which revealed a significant positive correlation between seminal fluid Chitotriosidase-1 activity and malondialdehyde levels (r = 0.37, P < 0.05) in the Asthenospermia Group. Conclusion: There is a correlation between seminal fluid Chitotriosidase-1 activity and malondialdehyde level in the Asthenospermia Group. Novel diagnostic and therapeutic approaches for the treatment of male infertility may result from our growing understanding of the roles played by Chitotriosidase-1 and malondialdehyde in male reproductive health.

BackgroundTo address knowledge gaps, this study aimed to investigate the involvement of inflammasomes in the etiology of azoospermia. This study focused on the gene expression of key inflammasome components, including NLR family pyrin domain containing 3 (NLRP-3), CASPASE-1, Interleukin-1β (IL-1β), Interleukin-18 (IL-18), NLR family CARD domain-containing protein 4/ice protease-activating factor (NLRC-4/IPAF), and Absent in melanoma 2 (AIM-2).MethodsWe analyzed gene expression in blood and testicular tissue from patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). Additionally, we compared IL-1β and IL-18 expression levels in seminal plasma samples using the Enzyme-Linked Immunosorbent Assay (ELISA) method. For comparison, blood samples from normospermic (NS) individuals were also genetically evaluated.ResultsOur results indicated significantly higher gene expression of inflammasome components in NOA patients than those in OA patients either in blood or in testicular tissue. Both azoospermic groups exhibited higher mRNA levels of inflammasome genes comparing with those from blood samples of NS men. Seminal plasma samples showed significantly increased levels of IL-1β and IL-18 in NOA patients compared to men with OA. The ROC curve analysis indicated strong and significant predictive power of IL-18, AIM-2 and NLRC-4/IPAF gene expression profiles between NOA vs. NS patients and NOA vs. OA.ConclusionsOur findings highlight the role of hidden chronic inflammation in azoospermia, particularly within the NOA group. This study provides a foundation for further detailed research, which could aid in the development of diagnostic panels to differentiate between various azoospermic groups.

Sexual dimorphism involves distinct anatomical, physiological, behavioral, and developmental differences between males and females of the same species, influenced by factors prior to conception and during early development. These sex-specific traits contribute to varied phenotypes and individual disease risks within and across generations and understanding them is essential in mammalian studies. Hormones, sex chromosomes, and imprinted genes drive this dimorphism, with over half of quantitative traits in wildtype mice showing sex-based variation. This review focuses on the impact of paternal non-genetic factors on sexual dimorphism. We synthesize current research on how paternal health before conception affects offspring phenotypes in a sex-specific manner, examining mechanisms such as DNA methylation, paternally imprinted genes, sperm RNA, and seminal plasma. Additionally, we explore how paternal influences indirectly shape offspring through maternal behavior, uterine environment, and placental changes, affecting males and females differently. We propose mechanisms modulating sexual dimorphism during development, underscoring the need for sex-specific documentation in animal studies.

The availability of mature broodstock that produces high-quality gametes is one of the vital issues with fish reproduction. The effects of multiple hormonal stimulations and continuous stripping of the same individual males on spermatozoa quality are unclear. Thus, this study aimed to investigate motility parameters and seminal plasma compositions of common carp sperm during out-of-spawning continuous stripping stimulated by multiple hormonal injections. Five mature males ∼3 years old, each approximately 1.7–2.3 kg bodyweight cultured in RAS at a stable temperature of 21 °C were used in this study. Fish were treated monthly (at the beginning of each month from August to October) with carp pituitary at 1.5 mg kg−1 (b.w.) dissolved in 0.9 % (w/v) NaCl solution 24 h before milt collection. The mean sperm concentration ranged from 17.55 to 20.19 × 109 mL−1 collected during the experiments and did not significantly vary among the sampling months (F = 2.352, p = 0.1374). However, the repeated measures ANOVA showed significant effects of sampling months (F = 25.24, p = 0.0001) and individual males (F = 7.50, p = 0.0001) on the motility parameters and seminal plasma compositions, but their interaction effects were not significant (F = 1.31, p = 0.079). One-way ANOVA revealed a significant difference in spermatozoa motility (F = 10.51, p = 0.0001) and pH of the seminal plasma (F = 90.00, p = 0.0001), but did not differ in curvilinear velocity (VCL), straight-line velocity (VSL), and ionic compositions (Na+, K+, Cl-, Ca2+, total protein, and osmolality) of the seminal plasma (p > 0.05). The mean spermatozoa motility in August (88.51–94.05 %) and September (89.88–96.00 %) was significantly higher than in October (83.99–89.58 %), but spermatozoa motility was >80 % in all males for the three consecutive months. The present study indicated that multiple hormonal stimulation and continuous stripping did not suppress common carp's spermatozoa production and quality parameters.

Abstract Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.

Abstract Aim The present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via natural mating, artificial insemination (AI), or intravaginal deposition (vaginal fundus) of different biological fluids or a placebo. Materials and methods In a double-blind study, six German Holstein cows were administered PGF2α during dioestrus and 48 h later treated with GnRH. Intravaginal or intrauterine treatments were carried out 12 h after GnRH was administered. Animals served as their controls, using a cross-over design with an interval of 14 days between experiments. The experimental animals were allocated to receive the following treatments: natural mating (N), intrauterine artificial insemination (A), intravaginal deposition (vaginal fundus) of 6 mL raw semen (R) or 6 mL seminal plasma (S), and compared to their controls [control 1: 6 mL placebo (P: physiological saline); control 2: no treatment (C)). Corresponding time intervals were chosen for the untreated control oestrus. Blood flow volume (BFV) in the uterine (u) and vaginal (v) arteries ipsilateral to the ovary bearing the preovulatory follicle was determined using transrectal Doppler sonography. Results All animals exhibited oestrus and ovulated between 30 and 36 h after GnRH. Transient increases (P 0.05). Only mating and deposition of either raw semen, seminal plasma or AI increased uBFV (P

This experiment conducted in the poultry fields, Department of Animal Production / College of Agriculture, University of Anbar from 1 December 2021 to 2 April 2022 in order to study the effect of semen storage process after centrifugation and replacement of seminal plasma with Lake (1960) diluent to preserve semen with the addition of the hormone melatonin as an antioxidant. This study conducted on 40 roosters of 33 weeks old Iraqi native rooster domestic chickens under standard conditions of temperature, humidity, ventilation and a balanced diet. Semen collecting samples divided into five treatments, three replicates for each one as follows. T1: control, T2: adding diluent as (1:2) semen: diluent only T3: adding a diluent containing the hormone melatonin at a concentration (1 mMol.L‾¹) T4: using Centrifuge with the addition of the dilution in the ratio (1:2) only T5: Use a centrifuge with the addition of a diluent containing the hormone melatonin at a concentration (1 mMol.L‾¹). Results showed that superiority in T3 treatment for seminal plasma traits, as it decreased significantly (P

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin. [ABSTRACT FROM AUTHOR]

Infertility affects a significant proportion of the reproductive‐aged population, with male‐associated factors contributing to over half of the cases. However, current diagnostic tools have limitations, leading to an underestimation of the true prevalence of male infertility. While traditional semen parameters provide some insights, they fail to determine the true fertility potential in a substantial number of instances. Therefore, it is crucial to investigate additional molecular targets responsible for male infertility to improve understanding and identification of such cases. Seminal plasma, the main carrier of molecules derived from male reproductive glands, plays a crucial role in reproduction. Amongst its multifarious functions, it regulates processes such as sperm capacitation, sperm protection and maturation, and even interaction with the egg's zona pellucida. Seminal plasma offers a non‐invasive sample for urogenital diagnostics and has shown promise in identifying biomarkers associated with male reproductive disorders. This review aims to provide an updated and comprehensive overview of seminal plasma in the diagnosis of male infertility, exploring its composition, function, methods used for analysis, and the application of emerging markers. Apart from the application, the potential challenges of seminal plasma analysis such as standardisation, marker interpretation and confounding factors have also been addressed. Moreover, we have also explored future avenues for enhancing its utility and its role in improving diagnostic strategies. Through comprehensive exploration of seminal plasma's diagnostic potential, the present analysis seeks to advance the understanding of male infertility and its effective management. [ABSTRACT FROM AUTHOR]

To detect whether intravaginal exposure to prepared seminal plasma led to an absolute increase in live birth rate (LBR) after in vitro fertilization (IVF) by 10% compared with placebo. It has been suggested that intravaginal deposition of seminal plasma after ovum pick-up (OPU) for IVF treatment, increases pregnancy and LBRs. Double-blind, placebo-controlled prospective study. An outcome assessment was made before the type of intervention was unblinded. The outcome data were analyzed according to an intention-to-treat protocol. University Hospital. Couples scheduled for an IVF treatment cycle: in total, 792 couples (393 in the seminal plasma group and 399 in the control group) were recruited over a 5-year period of inclusion in a single-center setting. On the day of OPU, the couples were randomized into groups receiving either vaginal deposition of prepared seminal plasma from the partner or saline. Both participants and the physician were blind to the grouping. The primary outcome was a live birth (LB). The secondary outcomes were a positive pregnancy test, defined as human chorionic gonadotropin identified in urine 3 weeks after OPU , and clinical pregnancy, defined as an intrauterine viable pregnancy assessed using transvaginal sonography after 5–7 weeks. In the index group, 35.4% had a positive pregnancy test (relative risk [RR],0.93; 95% confidence interval {CI} 0.78–1.10), 28.8% had a clinical pregnancy (RR 1.00, 95% CI 0.97–1.03), and 26.5% had a LB (RR 0.86; 95% CI 0.70–1.07), adjusted for day of transfer, female age, and number of fertilized oocytes. Corresponding rates in the control group were 37.3%, 33.6%, and 29.8%. No statistically significant differences regarding outcomes between the two intervention groups were found. Prepared seminal plasma applied in the vagina directly after OPU did not increase the rates of LB or clinical pregnancies. The importance of immunological factors to allow the implantation of an embryo is not questioned, but no improvement in the LBRs in IVF treatment by introducing the male partner's prepared seminal plasma after OPU could be found. Clinicaltrials.gov, ID NCT02716753. Registration date 17 March, 2016, first enrollment November, 2016, completed March, 2023. [ABSTRACT FROM AUTHOR]

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography–high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality. [ABSTRACT FROM AUTHOR]

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T‐cells. In a mixed lymphocyte reaction, T‐cell proliferation was inhibited by spEVs. A direct effect of spEVs on T‐cells was demonstrated when isolated T cells were activated by anti‐CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN‐γ, TNF, and IL‐2. Moreover, spEVs stimulated the expression of Foxp3 and IL‐10 by CD4+CD25+CD127‐ T cells, indicating differentiation into regulatory T‐cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T‐cells, indicating a requirement for surface‐exposed spEV proteins. The adenosine A2A receptor‐specific antagonist CPI‐444 also reduced effects of spEVs on T‐cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine‐A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T‐cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome‐localized A2A receptor signalling in spEVs targeted T‐cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs. [ABSTRACT FROM AUTHOR]

Aim: The present study was performed to characterize and compare the perfusion of vaginal and uterine arteries after challenging the reproductive tract of dairy cows via natural mating, artificial insemination (AI), or intravaginal deposition (vaginal fundus) of different biological fluids or a placebo. Materials and methods: In a double-blind study, six German Holstein cows were administered PGF2α during dioestrus and 48 h later treated with GnRH. Intravaginal or intrauterine treatments were carried out 12 h after GnRH was administered. Animals served as their controls, using a cross-over design with an interval of 14 days between experiments. The experimental animals were allocated to receive the following treatments: natural mating (N), intrauterine artificial insemination (A), intravaginal deposition (vaginal fundus) of 6 mL raw semen (R) or 6 mL seminal plasma (S), and compared to their controls [control 1: 6 mL placebo (P: physiological saline); control 2: no treatment (C)). Corresponding time intervals were chosen for the untreated control oestrus. Blood flow volume (BFV) in the uterine (u) and vaginal (v) arteries ipsilateral to the ovary bearing the preovulatory follicle was determined using transrectal Doppler sonography. Results: All animals exhibited oestrus and ovulated between 30 and 36 h after GnRH. Transient increases (P < 0.05) in vaginal blood flow occurred between 3 and 12 h following mating as well as 3 to 9 h after deposition of raw semen and seminal plasma, respectively. The most distinct increases (199%) in vBFV occurred 6 h after mating compared to values immediately before mating (= time 0 h). Neither AI nor deposition of a placebo into the vagina affected vBFV (P > 0.05). Only mating and deposition of either raw semen, seminal plasma or AI increased uBFV (P < 0.003). The greatest rise in uBFV occurred after natural mating. Maximum uBFV values were detected 9 h after mating when values were 79% greater (P < 0.05) than at 0 h. Conclusions: The natural mating, deposition of raw semen or seminal plasma and conventional AI affect vaginal and/or uterine blood flow to different degrees. The factors responsible for these alterations in blood flow and their effects on fertility remain to be clarified in future studies. [ABSTRACT FROM AUTHOR]

Background: Despite the clear clinical diagnostic criteria for necrozoospermia in andrology, the fundamental mechanisms underlying it remain elusive. This study aims to profile the lipid composition in seminal plasma systematically and to ascertain the potential of lipid biomarkers in the accurate diagnosis of necrozoospermia. It also evaluates the efficacy of a lipidomics-based random forest algorithm model in identifying necrozoospermia. Methods: Seminal plasma samples were collected from patients diagnosed with necrozoospermia (n = 28) and normozoospermia (n = 28). Liquid chromatography–mass spectrometry (LC–MS) was used to perform lipidomic analysis and identify the underlying biomarkers. A lipid functional enrichment analysis was conducted using the LION lipid ontology database. The top 100 differentially significant lipids were subjected to lipid biomarker examination through random forest machine learning model. Results: Lipidomic analysis identified 46 lipid classes comprising 1267 lipid metabolites in seminal plasma. The top five enriched lipid functions as follows: fatty acid (FA) with ≤ 18 carbons, FA with 16–18 carbons, monounsaturated FA, FA with 18 carbons, and FA with 16 carbons. The top 100 differentially significant lipids were subjected to machine learning analysis and identified 20 feature lipids. The random forest model identified lipids with an area under the curve > 0.8, including LPE(20:4) and TG(4:0_14:1_16:0). Conclusions: LPE(20:4) and TG(4:0_14:1_16:0), were identified as differential lipids for necrozoospermia. Seminal plasma lipidomic analysis could provide valuable biochemical information for the diagnosis of necrozoospermia, and its combination with conventional sperm analysis may improve the accuracy and reliability of the diagnosis. [ABSTRACT FROM AUTHOR]

This study aimed to determine whether male stress is related to seminal stress biomarkers and pregnancy achievement in women exposed to their partner's seminal plasma (SP) in the intracytoplasmic sperm injection (ICSI) cycle. In this pilot prospective study, 20 couples undergoing ICSI, as well as 5 fertile sperm donors and 10 saliva donors, were investigated. Women were exposed to their partner's SP via unprotected sexual intercourse during the ICSI cycle and intravaginal application on the day of ovum pick-up (Day-OPU). Semen samples were collected from male partners by masturbation on the Day-OPU. Saliva and serum samples were collected prior to masturbation. Body fluids were frozen at − 80 °C until assayed. Biomarkers of activity of the sympathetic adrenomedullary axis (salivary alpha-amylase and adrenaline), sympathetic neural axis (noradrenaline and dopamine), hypothalamic–pituitary–adrenal (HPA) system (cortisol), and immune system (C-reactive protein and interleukin (IL)-18) were estimated to examine their association with SP composition and clinical pregnancy achievement. The clinical pregnancy rate was 45.0%. In the unsuccessful ICSI group, blunted levels of salivary and serum cortisol were found compared to the successful ICSI group and the fertile sperm donors. With regard to seminal markers, decreased cortisol level and elevated noradrenaline, noradrenaline/cortisol ratio, and lL-18 levels were strongly associated with ICSI failure (areas under the ROC curves were, 0.813, 0.848, 0.899, and 0.828, respectively). These findings confirm that stress response systems activity affects SP composition, which in turn is associated with ICSI outcomes in women exposed to their partner's SP during an ICSI cycle. [ABSTRACT FROM AUTHOR]

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BACKGROUND The last decade has seen increased research on the relationship between diet and male fertility, but there are no clearly defined nutritional recommendations for men in the preconception period to support clinical fertility outcomes. OBJECTIVE AND RATIONALE The purpose of this scoping review is to examine the extent and range of research undertaken to evaluate the effect(s) of diet in the preconception period on male clinical fertility and reproductive outcomes. SEARCH METHODS Four electronic databases (MEDLINE and EMBASE via Ovid, CAB Direct, and CINAHL via EBSCO) were searched from inception to July 2023 for randomized controlled trials (RCTs) and observational studies (prospective/retrospective, case–control, and cross-sectional). Intervention studies in male participants or couples aiming to achieve dietary or nutritional change, or non-intervention studies examining dietary or nutritional components (whole diets, dietary patterns, food groups or individual foods) in the preconception period were included. Controls were defined as any comparison group for RCTs, and any/no comparison for observational studies. Primary outcomes of interest included the effect(s) of male preconception diet on clinical outcomes such as conception (natural or via ART), pregnancy rates and live birth rates. Secondary outcomes included time to conception and sperm parameters. OUTCOMES A total of 37 studies were eligible, including one RCT and 36 observational studies (prospective, cross-sectional, and case–control studies; four studies in non-ART populations) published between 2008 and 2023. Eight reported clinical outcomes, 26 reported on secondary outcomes, and three reported on both. The RCT did not assess clinical outcomes but found that tomato juice may benefit sperm motility. In observational studies, some evidence suggested that increasing fish or reducing sugar-sweetened beverages, processed meat or total fat may improve fecundability. Evidence for other clinical outcomes, such as pregnancy rates or live birth rates, showed no relationship with cereals, soy and dairy, and inconsistent relationships with consuming red meat or a 'healthy diet' pattern. For improved sperm parameters, limited evidence supported increasing fish, fats/fatty acids, carbohydrates and dairy, and reducing processed meat, while the evidence for fruits, vegetables, cereals, legumes, eggs, red meat and protein was inconsistent. Healthy diet patterns in general were shown to improve sperm health. WIDER IMPLICATIONS Specific dietary recommendations for improving male fertility are precluded by the lack of reporting on clinical pregnancy outcomes, heterogeneity of the available literature and the paucity of RCTs to determine causation or to rule out reverse causation. There may be some benefit from increasing fish, adopting a healthy dietary pattern, and reducing consumption of sugar-sweetened beverages and processed meat, but it is unclear whether these benefits extend beyond sperm parameters to improve clinical fertility. More studies exploring whole diets rather than singular foods or nutritional components in the context of male fertility are encouraged, particularly by means of RCTs where feasible. Further assessment of core fertility outcomes is warranted and requires careful planning in high-quality prospective studies and RCTs. These studies can lay the groundwork for targeted dietary guidelines and enhance the prospects of successful fertility outcomes for men in the preconception period. Systematic search of preconception diet suggests that increasing fish and reducing sugary drinks, processed meats and total fat may improve male fertility, while consuming healthy diets, fish, fats/fatty acids, carbohydrates and dairy and reducing processed meat can improve sperm health. [ABSTRACT FROM AUTHOR]

The investigation of biomarkers is constantly evolving. New molecules and molecular assemblies, such as soluble and particulate complexes, emerged as biomarkers from basic research and investigation of different proteomes, genomes, and glycomes. Extracellular vesicles (EVs), and glycans, complex carbohydrates are ubiquitous in nature. The composition and structure of both reflect physiological state of paternal cells and are strikingly changed in diseases. The EV-associated glycans, alone or in combination with soluble glycans in related biological fluids, used as analytes, aim to capture full complex biomarker picture, enabling its use in different clinical settings. Bringing together EVs and glycans can help to extract meaningful data from their extreme and distinct heterogeneities for use in the real-time diagnostics. The glycans on the surface of EVs could mark their subpopulations and establish the glycosignature, the solubilisation signature and molecular patterns. They all contribute to a new way of looking at and looking for composite biomarkers. [ABSTRACT FROM AUTHOR]

Male infertility is a global public health issue, but studies on the correlation between the dietary components and sperm quality showed inconclusive results due to the heterogeneous population with different dietary habits and environmental stimuli. Herein, the correlation of dietary macro- and micro-mineral intake was evaluated with quality/quantity and oxidant/antioxidant status of seminal fluid in infertile compared to the healthy men. One hundred twenty men attending to the infertility clinic of Ayatollah Mousavi Hospital in Zanjan City were enrolled. Seminal fluid was extracted, and groups were categorized into the infertile (non-standard) and normal (standard) groups based on the WHO, 2020 criteria. Food frequency questionnaire was completed. Seminal malondialdehyde (MDA) and total antioxidant capacity (TAC) were measured by ELISA kit based on the manufacture's instruction. An independent sample t-test was used to determine differences between the two groups, and linear regression model was used to determine the effect of each dietary macro/micro mineral intake on these parameters. Adjusting for all parameters, dietary selenium increased 3.7-folds the seminal TAC level (p=0.04) and decreased sperm with non-progressive motility by 2.4-folds (p=0.04). Higher manganese intake increased the sperm count by 7.8-folds (p=0.005). Dietary copper decreased sperm vitality and increased sperm with slow motility (OR= −1.7, 95% CI= −59.8, −9.9; p=0.007). Dietary zinc (OR=1.24, p=0.01) and iron (OR=1.5, p=0.02) showed a positive effect on sperm vitality. None of macro and micro minerals showed a significant effect on the seminal MDA level. Daily intake of adequate amounts of micro and macro minerals improves sperm quality and increases the antioxidant capacity of the seminal fluid; however, copper showed a negative correlation that must be evaluated in future studies. [ABSTRACT FROM AUTHOR]